Project Summary Wolbachia offer a pesticide-free alternative to curb arbovirus transmission. Current pilot studies release infected mosquitoes to either crash the resident population size or spread mosquitoes with reduced vectorial capacity in the local population. Both efforts rely upon Wolbachia's adaptation to cause cytoplasmic incompatibility (CI) - a sperm modification that causes embryonic arrest in crosses between infected males and uninfected females (unidirectional CI) or between males and females harboring different Wolbachia strains (bidirectional CI). However, infected females with the same strain rescue the sperm modification, giving them a large fitness advantage over uninfected females that in turn enables rapid spread of Wolbachia through host populations. Despite four decades of intense research and current applications to vector control, the underlying genetic basis of CI has not been reported. However, we recently identified two prophage WO gene candidates in the released strain of wMel Wolbachia that natively infects Drosophila melanogaster. These genes are hereafter referred to as cytoplasmic incompatibility factor A (cifA) and cytoplasmic incompatibility factor B (cifB). cifA and cifB exhibit enhanced expression in infected host testes, as expected for genes involved in CI. Expression of cifA and cifB transgenes in uninfected D. melanogaster testes recapitulates induction of CI, while expression in Wolbachia- infected testes enhances the wild type CI. Importantly, wMel-infected females rescue the transgene-associated CI, thereby implicating these candidates as likely CI genes. Phylogenies specify divergent clades of cifA and cifB homologs that covary with bidirectional incompatibility relationships between Wolbachia strains. Additionally, recent genome sequencing and comparisons reveal candidate rescue genes that exhibit parallel phylogenies to those of cifA and cifB and preferential expression in early embryos. As these findings represent a potential breakthrough in the search for CI genes, the central hypothesis of the proposed research is that the candidate modification and rescue genes determine unidirectional and bidirectional CI. In Aim 1, we will use transgenic expression of genetically divergent cifA and cifB variants to broadly test the essential cif gene regions for inducing CI and whether extant homologs of these genes retain the ability to induce CI. In Aim 2, we will investigate candidate rescue genes from wMel using transgenic expression in females and crosses with an array of males with CI-inducing Wolbachia or transgene cif homologs. This aim will also explicitly test the presumption that divergence in modification and rescue genes underlies bidirectional CI. Examinations thus far have yet to yield a comprehensive genetic advance for both sides of CI, and the rising interest in deploying Wolbachia to curb arbovirus transmission necessitates an explanation of Wolbachia's drive system. If successful, this research will pioneer genetic studies of bacteria-induced reproductive parasitism, inform Wolbachia's efficacy and delivery as a tool to control diverse zoonotic diseases, and provide multiple lines of evidence for the discovery of genes that underlie CI.